This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes of the carotenoid biosynthesis pathway in plants and seeds.
Plant carotenoids are orange and red lipid-soluble pigments found embedded in the membranes of chloroplasts and chromoplasts. In leaves and immature fruits the color is masked by chlorophyll but in later stages of development these pigments contribute to the bright color of flowers and fruits. Carotenoids protect against photoxidation processes and harvest light for photosynthesis. The carotenoid biosynthesis pathway leads to the production of abscisic acid with intermediaries useful in the agricultural and food industries as well as products thought to be involved in cancer prevention. (Bartley, G. E., and Scolnik, P. A. (1995) Plant Cell 7:1027-1038).
Phytoene synthase carries out the first step in the carotenoid biosynthetic pathway converting geranylgeranyl diphosphate to phytoene. There are two different phytoene synthases in tomato with different expression patterns: one is expressed at higher levels in mature fruits while the other one is expressed at higher levels in leaves (Bartley, G. E., Scolnik, P. A. (1993) J. Biol Chem. 268:25718-25721). It has been speculated that in corn at least two different alleles of phytoene synthase should be present but only one has been identified to date (Buckner, B. et al. (1996) Genetics 143:479-488).
In the next step of the carotenoid biosynthesis pathway, phytoene desaturase transforms phytoene into phytofluene. After another desaturation step, the enzyme zeta-carotene desaturase (carotene 7, 8 desaturase; EC 1.134.99.30) converts the lightly colored zeta-carotene to neurosporene which is further desaturated into lycopene. Lycopene may have one of two different fates: through the action of lycopene epsilon cyclase it may become alpha carotene, or it may be transformed into beta carotene by lycopene cyclase. Beta-carotene dehydroxylase converts beta-carotene into zeaxanthin. Zeaxanthin epoxidase transforms zeaxanthin into violxanthin and eventually abscisic acid. The genes encoding this chloroplast-imported protein have been identified in N. plumbaginifolia, pepper and tomato. Zeaxanthin epoxidase appears to also be involved in protection from environmental stress (Corinne A. et al. (1998) Plant Phys. 118:1021-1028) and uses FAD as a cofactor (Buch, K. et al. (1995) FEBS Lett. 376:45-48).
Zeaxanthin is the bright orange product highly prized as a pigmenting agent for animal feed which makes the meat fat, skin, and egg yolks a dark yellow (Scott, M. L. et al. (1968) Poultry Sci. 47:863-872). Gram per gram, zeaxanthin is one of the best pigmenting compounds because it is highly absorbable. Yellow corn, which produces one of the best ratios of lutein to zeaxanthin contains in average 20 to 25 mg of xanthophyll per kg while marigold petals yield 6,000 to 10,000 mg/kg.
The instant invention relates to isolated nucleic acid fragments encoding carotenoid biosynthetic enzymes. Specifically, this invention concerns an isolated nucleic acid fragment encoding a phytoene synthase or a zeaxanthin epoxidase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding phytoene synthase or zeaxanthin epoxidase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a carotenoid biosynthetic enzyme selected from the group consisting of phytoene synthase and zeaxanthin epoxidase.
In another embodiment, the instant invention relates to a chimeric gene encoding a phytoene synthase or a zeaxanthin epoxidase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a phytoene synthase or a zeaxanthin epoxidase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a phytoene synthase or a zeaxanthin epoxidase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a phytoene synthase or a zeaxanthin epoxidase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a phytoene synthase or a zeaxanthin epoxidase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of phytoene synthase or zeaxanthin epoxidase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a phytoene synthase or a zeaxanthin epoxidase.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a phytoene synthase or a zeaxanthin epoxidase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a phytoene synthase or a zeaxanthin epoxidase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of phytoene synthase or zeaxanthin epoxidase in the transformed host cell; (c) optionally purifying the phytoene synthase or the zeaxanthin epoxidase expressed by the transformed host cell; (d) treating the phytoene synthase or the zeaxanthin epoxidase with a compound to be tested; and (e) comparing the activity of the phytoene synthase or the zeaxanthin epoxidase that has been treated with a test compound to the activity of an untreated phytoene synthase or zeaxanthin epoxidase, thereby selecting compounds with potential for inhibitory activity.